The brains of patients with Leigh's disease are deficient in thiamine triphosphate and contain an excess of thiamine pyrophosphate. All body fluids of patients, as well as obligate carriers of the Leigh's disease gene, contain a protein which blocks the synthesis of thiamine triphosphate by a cerebral microsomal enzyme. We propose to isolate this protein from the urine of untreated patients as well as their parents, and to purify the brain enzyme whose activity it inhibits. When these purifications are complete we should be able to: (1) identify the molecular structure of the inhibitor, (2) describe the dynamics of the inhibition, (3) reproduce the disease by injecting the inhibitor into growing laboratory animals, and (4) demonstrate why thiamine therapy may have a therapeutic benefit for patients. The second aim of this research proposal is to identify the relationship between hexosaminidase A and hexosaminidase B. We have shown that thex A can be converted to an enzyme behaving like hex B by the action of a cerebral neuroaminidase. We propose to study the reverse reaction: i.e., the conversion of hex B to hex A by the addition of NANA to hex B. The specific hexosaminidase isozymes will be identified by their action on the natural substrate.